1. Field of the Invention
The present invention comprises devices and methods that use optical microcavities as a spectroscopic tool for detecting molecules and for probing conformations and orientations of molecules and molecular assemblies.
2. Brief Description of the Related Art
Optical microresonators with small modal volumes and high quality (Q) factors significantly enhance interaction of the optical field with the material through recirculation, which makes them exceptionally sensitive to the optical properties of the resonator and the surrounding medium. (See, for example, Spillane, S. M., T. J. Kippenberg, and K. J. Vahala, “Ultralow-threshold Raman laser using a spherical dielectric microcavity,” Nature 415:621-623 (2002); V. R. Almeida, C. A. Barrios, R. R. Panepucci, M. Lipson, “All-optical control of light on a silicon chip,” Nature 431, 1081-1084 (2004); V. S. Ilchenko, A. A. Savchenkov, A. B. Matsko, L. Maleki, “Nonlinear optics and crystalline whispering gallery mode cavities,” Phys. Rev. Lett. 92:043903 (2004); and E. Krioukov, D. J. W. Klunder, A. Driessen, J. Greve, C. Otto, “Sensor based on an integrated optical microcavity,” Opt. Lett. 27, 512 (2002).)
Exploiting this attribute, optical microcavities have been used successfully for ultra-sensitive detection of heavy water and biomolecules. (See A. M. Armani, K. J. Vahala, “Heavy water detection using ultra-high-Q microcavities,” Opt. Lett. 31:1896-1898 (2006); F. Vollmer, D. Braun, A. Libchaber, M. Khoshsima, I. Teraoka, S. Arnold, “Protein detection by optical shift of a resonant microcavity,” Appl. Phys. Lett. 80:4057-4059 (2002); F. Vollmer, S. Arnold, D. Braun, I. Teraoka, A. Libchaber, “Multiplexed DNA quantification by spectroscopic shift of two microsphere cavities,” Biophys. J. 85:1974-1979 (2003); M. Noto, F. Vollmer, D. Keng, I. Teraoka, S. Arnold, “Nanolayer characterization through wavelength multiplexing of a microsphere resonator,” Opt. Lett. 30, 510 (2005).) In biosensing, the sensitivity of microcavities surpasses that of surface-plasmon resonance (SPR) which is widely recognized as the state-of-the-art label-free detection technique. Binding of only a few molecules on the microcavity surface shifts the frequencies of the resonant modes that evanescently interact with the adsorbed material. Monitoring of the shift induced by polarizability or refractive index changes forms the basis of label-free, non-invasive, real-time biodetection and nanolayer characterization. The present invention extends this sensitive technique using devices and methods for pump-probe spectroscopy in high-Q microcavities which can be used to detect molecules and to track dynamic changes in the molecular structure.
Coupled plasmon-waveguide resonance (CPWR) spectroscopy has been developed to probe anisotropies in biological membranes immobilized onto solid surfaces by incorporating a TM- (transverse magnetic) polarized probe in addition to the TE- (transverse electric) polarized one used in conventional SPR. (See Z. Salamon, H. A. Macleod, G. Tollin, “Coupled plasmon-waveguide resonators: A new spectroscopic tool for probing proteolipid film structure and properties,” Biophys. J. 73: 2791-2797 (1997).) High-Q optical microcavities represent an interesting alternative to SPR-based techniques providing exceptional sensitivity and two possible probing polarizations and operation at arbitrary wavelengths.
The magnitude of the polarizability changes (A) that accompany structural transformations in, e.g., complex proteolipid macromolecules, is not known, although conformational changes in such systems have been observed qualitatively by CPWR or indirectly by Stark spectroscopy. Given their superior sensitivity and the ability to directly quantify Δα, the microresonators represent a new quantitative tool for probing molecular transformations in important proteolipid biomolecular assemblies such as G-protein-coupled receptors or in signaling molecules such as calmodulin and disease-related prion proteins. As disclosed herein for the model case of Bacteriorhodopsin (bR), the present invention is particularly suited for pump-probe studies of photosensitive biomolecules. Further examples of such systems include photosynthetic membranes and photoreceptors such as photoactive yellow protein. Further improvements of the microresonator technique promise single-molecule sensitivity which is beyond the scope of any alternative label-free technique. (See S. Arnold, M. Khoshsima, I. Teraoka, S. Holler, F. Vollmer, “Shift of whispering-gallery modes in microspheres by protein adsorption,” Opt. Lett. 28:272-274 (2003).) For such applications, a pump-probe approach, where a probe as well as a pump beam excites a cavity resonance, provides added sensitivity for detection of single particles and molecules since absorption of a pump-beam by molecules/particles generates heat, the effect of which can be measured with a probe due to thermally induced refractive index changes. The thermo-optic effect has been demonstrated in toroidal microcavities utilizing only one beam at resonance with a microcavity (A. M. Armani*, R. P. Kulkarni, S. E. Fraser, R. C. Flagan, K. J. Vahala “Label-free, single-molecule detection with optical microcavities” Science, Volume 317, 5839, August 2007). The present invention utilizes a pump probe approach where the resonance wavelength of the pump beam can be chosen deliberately to maximize absorption by molecular analytes and particles. Although high circulating power is advantageous, the resonance excited by the pump beam does not have to be associated with a high-Q factor, since sensitive detection is achieved with a probe beam that (simultaneously or sequentially) excites a high-Q resonance in the same microcavity. The resonance wavelength of the probe beam is chosen to maximize Q-factor and thus sensitivity for detection.